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International audience; Diatoms are successful phytoplankton clades able to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity via the nonphotochemical quenching (NPQ) process. While the molecular effectors of NPQ as well as the involvement of the proton motive force (PMF) in its regulation are known, the regulators of the PET/PMF relationship remain unidentified in diatoms. We generated mutants of the H$^+$ /K$^+$ antiporter KEA3 in the model diatom $Phaeodactylum\ tricornutum$. Loss of KEA3 activity affects the PET/PMF coupling and NPQ responses at the onset of illumination, during transients and in steady-state conditions. Thus, this antiporter is a main regulator of the PET/PMF coupling. Consistent with this conclusion, a parsimonious model including only two free components, KEA3 and the diadinoxanthin de-epoxidase, describes most of the feedback loops between PET and NPQ. This simple regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory calcium ion (Ca$^{2+}$ )-binding domain in KEA3 modulating its activity. This circuit is likely tuned by the NPQ-effector proteins, LHCXs, providing diatoms with the required flexibility to thrive in different ocean provinces.

International audience; Aminoglycosides (AG) have been used against Gram-negative bacteria for decades. Yet, how bacterial metabolism and environmental conditions modify AG toxicity is poorly understood. Here, we show that the level of AG susceptibility varies depending on the nature of the respiratory chain that Escherichia coli uses for growth, i.e., oxygen, nitrate, or fumarate. We show that all components of the fumarate respiratory chain, namely, hydrogenases 2 and 3, the formate hydrogenlyase complex, menaquinone, and fumarate reductase are required for AG-mediated killing under fumarate respiratory conditions. In addition, we show that the AAA+ ATPase RavA and its Von Wildebrand domain-containing partner, ViaA, are essential for AG to act under fumarate respiratory conditions. This effect was true for all AG that were tested but not for antibiotics from other classes. In addition, we show that the sensitizing effect of RavA-ViaA is due to increased gentamicin uptake in a proton motive force-dependent manner. Interestingly, the sensitizing effect of RavA-ViaA was prominent in poor energy conservation conditions, i.e., with fumarate, but dispensable under high energy conservation conditions, i.e., in the presence of nitrate or oxygen. We propose that RavA-ViaA can facilitate uptake of AG across the membrane in low-energy cellular states. IMPORTANCE Antibiotic resistance is a major public health, social, and economic problem. Aminoglycosides (AG) are known to be highly effective against Gram-negative bacteria, but their use is limited to life-threatening infections because of their nephrotoxicity and ototoxicity at therapeutic dose. Elucidation of AG-sensitization mechanisms in bacteria would allow reduced effective doses of AG. Here, we have identified the molecular components involved in anaerobic fumarate respiration that are required for AG to kill. In addition to oxidoreductases and menaquinone, this includes new molecular players, RavA, an AAA+ ATPase, and ViaA, its partner that has the VWA motif. Remarkably, the influence of RavA-ViaA on AG susceptibility varies according to the type of bioenergetic metabolism used by E. coli. This is a significant advance because anaerobiosis is well known to reduce the antibacterial activity of AG. This study highlights the critical importance of the relationship between culture conditions, metabolism, and antibiotic susceptibility.

Bacterial flagellar subunits are exported across the cell membrane by the flagellar Type III Secretion System (fT3SS), powered by the proton motive force (pmf) and a specialized ATPase that enables the flagellar export gate to utilize the pmf electric potential (ΔΨ). Export gate activation is mediated by the ATPase stalk, FliJ, but how this process is regulated to prevent wasteful dissipation of pmf in the absence of subunit cargo is not known. Here, we show that FliJ activation of the export gate is regulated by flagellar export chaperones. FliJ binds unladen chaperones and, by using novel chaperone variants specifically defective for FliJ binding, we show that disruption of this interaction attenuates motility and cognate subunit export. We demonstrate in vitro that chaperones and the FlhA export gate component compete for binding to FliJ, and show in vivo that unladen chaperones, which would be present in the cell when subunit levels are low, sequester FliJ to prevent activation of the export gate and attenuate subunit export. Our data indicate a mechanism whereby chaperones couple availability of subunit cargo to pmf‐driven export by the fT3SS.

Driven by transmembrane electrochemical ion gradients, F-type ATP synthases are the primary source of the universal energy currency, adenosine triphosphate (ATP), throughout all domains of life. The ATP synthase found in the thylakoid membranes of photosynthetic organisms has some unique features not present in other bacterial or mitochondrial systems. Among these is a larger-than-average transmembrane rotor ring and a redox-regulated switch capable of inhibiting ATP hydrolysis activity in the dark by uniquely adapted rotor subunit modifications. Here, we review recent insights into the structure and mechanism of ATP synthases specifically involved in photosynthesis and explore the cellular physiological consequences of these adaptations at short and long time scales.

Significance The newly described type IX secretion systems (T9SSs) translocate virulence factors and can mediate specialized gliding motility among bacterial pathogens of the Fibrobacteres–Chlorobi–Bacterioidetes superphylum. We visualized the spatial organization of the T9SS in its native context in the Porphorymonas gingivalis cell by cryoelectron tomography. The T9SS exhibits distinct symmetries across the bacterial cell envelope: a cytoplasmic complex requiring PorL and PorM for assembly exhibits 12-fold symmetry; a periplasmic complex composed of PorM exhibits 18-fold symmetry and attaches to a PorKN ring near the outer membrane; and eight Sov translocons are arranged with 8-fold symmetry at the cell surface. The T9SS is the largest of the known bacterial secretion systems and evidently arranges as multiple, independently functioning translocation motors.

Cobamides are cobalt-containing cyclic tetrapyrroles involved in the metabolism of organisms from all domains of life but produced de novo only by some bacteria and archaea. The pathway is thought to involve up to 30 enzymes, five of which comprise the so-called “late” steps of cobamide biosynthesis. Two of these reactions activate the corrin ring, one activates the nucleobase, a fourth one condenses activated precursors, and a phosphatase yields the final product of the pathway. The penultimate step is catalyzed by a polytopic integral membrane protein, namely, the cobamide (5′-phosphate) synthase, also known as cobamide synthase. At present, the reason for the association of all putative and bona fide cobamide synthases to cell membranes is unclear and intriguing. Here, we show that, in Escherichia coli, elevated levels of cobamide synthase kill the cell by dissipating the proton motive force and compromising membrane stability. We also show that overproduction of the phosphatase that catalyzes the last step of the pathway or phage shock protein A prevents cell death when the gene encoding cobamide synthase is overexpressed. We propose that in E. coli, and probably all cobamide producers, cobamide synthase anchors a multienzyme complex responsible for the assembly of vitamin B12 and other cobamides.

Multidrug and toxic compound extrusion (MATE) transporters in eukaryotes have been characterized to be antiporters that mediate the transport of substrates in exchange for protons. In plants, alkaloids, phytohormones, ion chelators, and flavonoids have been reported to be the substrates of MATE transporters. Structural analyses have been conducted to dissect the functional significance of various motifs of MATE proteins. However, an understanding of the functions of the N- and C-termini has been inadequate. Here, by performing phylogenetic analyses and protein sequence alignment of 14 representative plant species, we identified a distinctive N-terminal poly-glutamate motif among a cluster of MATE proteins in soybean. Amongst them, GmMATE4 has the most consecutive glutamate residues at the N-terminus. A subcellular localization study showed that GmMATE4 was localized at the vacuolar membrane-like structure. Protein charge prediction showed that the mutation of the glutamate residues to alanine would reduce the negative charge at the N-terminus. Using yeast as the model, we showed that GmMATE4 mediated the transport of daidzein, genistein, glycitein, and glycitin. In addition, the glutamate-to-alanine mutation reduced the isoflavone transport capacity of GmMATE4. Altogether, we demonstrated GmMATE4 as an isoflavone transporter and the functional significance of the N-terminal poly-glutamate motif of GmMATE4 for regulating the isoflavone transport activity.

The classical view of oxidative phosphorylation is that a proton motive force (PMF) generated by the respiratory chain complexes fuels ATP synthesis via ATP synthase. Yet, under glycolytic conditions, ATP synthase in its reverse mode also can contribute to the PMF. Here, we dissected these two functions of ATP synthase and the role of its inhibitory factor 1 (IF1) under different metabolic conditions. pH profiles of mitochondrial sub‐compartments were recorded with high spatial resolution in live mammalian cells by positioning a pH sensor directly at ATP synthase’s F1 and FO subunits, complex IV and in the matrix. Our results clearly show that ATP synthase activity substantially controls the PMF and that IF1 is essential under OXPHOS conditions to prevent reverse ATP synthase activity due to an almost negligible ΔpH. In addition, we show how this changes lateral, transmembrane, and radial pH gradients in glycolytic and respiratory cells., Mitochondrial F1FO ATP synthase synthesizes or hydrolyses ATP depending on the metabolic conditions and controlled by IF1. Analysis of pH values using local pH sensors at ATP synthase and ATP synthase subcomplexes discloses these activities, which essentially contribute to the establishment of radial, transmembrane, and lateral pH gradients in cristae.

SummaryThe responses of plant photosynthesis to rapid fluctuations in environmental conditions are thought to be critical for efficient capture of light energy. Such responses are not well represented under laboratory conditions, but have also been difficult to probe in complex field environments. We demonstrate an open science approach to this problem that combines multifaceted measurements of photosynthesis and environmental conditions, and an unsupervised statistical clustering approach. In a selected set of data on mint (Menthasp.), we show that the “light potential” for increasing linear electron flow (LEF) and nonphotochemical quenching (NPQ) upon rapid light increases are strongly suppressed in leaves previously exposed to low ambient PAR or low leaf temperatures, factors that can act both independently and cooperatively. Further analyses allowed us to test specific mechanisms. With decreasing leaf temperature or PAR, limitations to photosynthesis during high light fluctuations shifted from rapidly-induced NPQ to photosynthetic control (PCON) of electron flow at the cytochromeb6fcomplex. At low temperatures, high light induced lumen acidification, but did not induce NPQ, leading to accumulation of reduced electron transfer intermediates, a situation likely to induce photodamage, and represents a potential target for improving the efficiency and robustness of photosynthesis. Finally, we discuss the implications of the approach for open science efforts to understand and improve crop productivity.

Protocatechuic acid (PCA) is a strong antioxidant and is also a potential platform for polymer building blocks like vanillic acid, vanillin, muconic acid, and adipic acid. This report presents a study on PCA production from glucose via the shikimate pathway precursor 3-dehydroshikimate by heterologous expression of a gene encoding 3-dehydroshikimate dehydratase in Escherichia coli. The phenylalanine overproducing E. coli strain, engineered to relieve the allosteric inhibition of 3-deoxy-7-phosphoheptulonate synthase by the aromatic amino acids, was shown to give a higher yield of PCA than the unmodified strain under aerobic conditions. Highest PCA yield of 18 mol% per mol glucose and concentration of 4.2 g/L was obtained at a productivity of 0.079 g/L/h during cultivation in fed-batch mode using a feed of glucose and ammonium salt. Acetate was formed as a major side-product indicating a shift to catabolic metabolism as a result of feedback inhibition of the enzymes including 3-dehydroshikimate dehydratase by PCA when reaching a critical concentration. Indirect measurement of proton motive force by flow cytometry revealed no membrane damage of the cells by PCA, which was thus ruled out as a cause for affecting PCA formation.

Oxidative phosphorylation is carried out by five complexes, which are the sites for electron transport and ATP synthesis. Among those, Complex V (also known as the F1F0ATP Synthase or ATPase) is responsible for the generation of ATP through phosphorylation of ADP by using electrochemical energy generated by proton gradient across the inner membrane of mitochondria. A multi subunit structure that works like a pump functions along the proton gradient across the membranes which not only results in ATP synthesis and breakdown, but also facilitates electron transport. Since ATP is the major energy currency in all living cells, its synthesis and function have widely been studied over the last few decades uncovering several aspects of ATP synthase. This review intends to summarize the structure, function and inhibition of the ATP synthase.

Significance The transmembrane electrical potential difference (Δψ), which is defined as membrane voltage, is used as the energy for many biological activities. For construction of the bacterial flagella on the cell surface, a specialized protein transporter utilizes Δψ to drive proton-coupled protein export, but it remains unknown how. Here, we report that an inactive flagellar protein transporter can be activated by an increase in Δψ above a threshold value through an interaction between FliJ and the transmembrane proton channel protein FlhA. Following activation, the protein transporter conducts protons through the FlhA channel to drive flagellar protein export. This report describes a Δψ-dependent activation mechanism used for a biological function other than voltage-gated ion channels., The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

Protocatechuic acid (PCA) is a strong antioxidant and is also a potential platform for polymer building blocks like vanillic acid, vanillin, muconic acid, and adipic acid. This report presents a study on PCA production from glucose via the shikimate pathway precursor 3-dehydroshikimate by heterologous expression of a gene encoding 3-dehydroshikimate dehydratase in Escherichia coli. The phenylalanine overproducing E. coli strain, engineered to relieve the allosteric inhibition of 3-deoxy-7-phosphoheptulonate synthase by the aromatic amino acids, was shown to give a higher yield of PCA than the unmodified strain under aerobic conditions. Highest PCA yield of 18 mol% per mol glucose and concentration of 4.2 g/L was obtained at a productivity of 0.079 g/L/h during cultivation in fed-batch mode using a feed of glucose and ammonium salt. Acetate was formed as a major side-product indicating a shift to catabolic metabolism as a result of feedback inhibition of the enzymes including 3-dehydroshikimate dehydratase by PCA when reaching a critical concentration. Indirect measurement of proton motive force by flow cytometry revealed no membrane damage of the cells by PCA, which was thus ruled out as a cause for affecting PCA formation.

Background In acidic soils, aluminum (Al) competing with Zn results in Zn deficiency in plants. Zn is essential for auxin biosynthesis. Zn-mediated alleviation of Al toxicity has been rarely studied, the mechanism of Zn alleviation on Al-induced photoinhibition in photosystems remains unclear. The objective of this study was to investigate the effects of Zn and IAA on photosystems of Al-stressed alfalfa. Alfalfa seedlings with or without apical buds were exposed to 0 or100 μM AlCl3 combined with 0 or 50 μM ZnCl2, and then foliar spray with water or 6 mg L− 1 IAA. Results Our results showed that Al stress significantly decreased plant growth rate, net photosynthetic rate (Pn), quantum yields and electron transfer rates of PSI and PSII. Exogenous application of Zn and IAA significantly alleviated the Al-induced negative effects on photosynthetic machinery, and an interaction of Zn and IAA played an important role in the alleviative effects. After removing apical buds of Al-stressed alfalfa seedlings, the values of pmf, gH+ and Y(II) under exogenous spraying IAA were significantly higher, and ΔpHpmf was significantly lower in Zn addition than Al treatment alone, but the changes did not occur under none spraying IAA. The interaction of Zn and IAA directly increased Y(I), Y(II), ETRI and ETRII, and decreased O2− content of Al-stressed seedlings. In addition, the transcriptome analysis showed that fourteen functionally noted genes classified into functional category of energy production and conversion were differentially expressed in leaves of alfalfa seedlings with and without apical buds. Conclusion Our results suggest that the interaction of zinc and IAA alleviate aluminum-induced damage on photosystems via increasing pmf and decreasing ΔpHpmf between lumen and stroma.

The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-motBfliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-motBfliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA&ndash, FliG interaction responsible for torque generation.

Vitamin B1 is a family of molecules, the most renowned member of which is diphosphorylated thiamine (TDP)—a coenzyme vital for the activity of key enzymes of energy metabolism. Triphosphorylated thiamine derivatives also exist within this family, specifically thiamine triphosphate (TTP) and adenosine thiamine triphosphate (ATTP). They have been investigated primarily in mammalian cells and are thought to act as metabolic messengers but have not received much attention in plants. In this study, we set out to examine for the presence of these triphosphorylated thiamine derivatives in Arabidopsis. We could find TTP in Arabidopsis under standard growth conditions, but we could not detect ATTP. Interestingly, TTP is found primarily in shoot tissue. Drivers of TTP synthesis are light intensity, the proton motive force, as well as TDP content. In plants, TTP accumulates in the organellar powerhouses, the plastids, and mitochondria. Furthermore, in contrast to other B1 vitamers, there are strong oscillations in tissue levels of TTP levels over diel periods peaking early during the light period. The elevation of TTP levels during the day appears to be coupled to a photosynthesis‐driven process. We propose that TTP may signify TDP sufficiency, particularly in the organellar powerhouses, and discuss our findings in relation to its role.

The objective of this study was to investigate the effects of Mg and IAA on the photosystems of Al-stressed alfalfa (Medicago sativa L.). Alfalfa seedlings with or without apical buds were exposed to solutions fully mixed with 0 or 100 μM AlCl3 and 0 or 50 μM MgCl2 followed by foliar spray with water or IAA. Results from seedlings with apical buds showed that application of Mg and IAA either alone or combine greatly alleviated the Al-induced damage on photosystems. The values of photosynthetic rate (Pn), effective quantum yields [Y(I) and Y(II)] and electron transfer rates (ETRI and ETRII), proton motive force (pmf), cyclic electron flow (CEF), proton efflux rate (gH +), and activities of ATP synthase and PM H+-ATPase significantly increased, and proton gradient (ΔpH pmf ) between lumen and stroma decreased under Al stress. After removing apical buds of seedlings, the Y(I), Y(II), ETRI, ETRII, pmf, and gH + under exogenous spraying IAA significantly increased, and ΔpH pmf significantly decreased in Mg addition than Al treatment alone, but they were no significant difference under none spraying IAA. The interaction of Mg and IAA directly increased quantum yields and electron transfer rates, and decreased O2 - accumulation in Al-stressed seedlings with or without apical buds. These results suggest that IAA involves in Mg alleviation of Al-induced photosystem damage via increasing pmf and PM H+-ATPase activity, and decreasing ΔpH pmf .

Acinetobacter baumannii, a Gram-negative opportunistic pathogen, is a leading cause of hospital- and community-acquired infections. Acinetobacter baumannii can rapidly acquire diverse resistance mechanisms and undergo genetic modifications that confer resistance and persistence to all currently used clinical antibiotics. In this study, we found exogenous L-lysine sensitizes Acinetobacter baumannii, other Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) and a Gram-positive bacterium (Mycobacterium smegmatis) to aminoglycosides. Importantly, the combination of L-lysine with aminoglycosides killed clinically isolated multidrug-resistant Acinetobacter baumannii and persister cells. The exogenous L-lysine can increase proton motive force via transmembrane chemical gradient, resulting in aminoglycoside acumination that further accounts for reactive oxygen species production. The combination of L-lysine and antibiotics highlights a promising strategy against bacterial infection.

The conversion of sunlight into useable cellular energy occurs via the proton-coupled electron transfer reactions of photosynthesis. Light is absorbed by photosynthetic pigments and transferred to photochemical reaction centers to initiate electron and proton transfer reactions to store energy in a redox gradient and an electrochemical proton gradient (proton motive force, pmf), composed of a concentration gradient (ΔpH) and an electric field (Δψ), which drives the synthesis of ATP through the thylakoid FoF1-ATP synthase. Although ATP synthase structure and function are conserved across biological kingdoms, the number of membrane-embedded ion-binding c subunits varies between organisms, ranging from 8 to 17, theoretically altering the H+/ATP ratio for different ATP synthase complexes, with profound implications for the bioenergetic processes of cellular metabolism. Of the known c-ring stoichiometries, photosynthetic c-rings are among the largest identified stoichiometries, and it has been proposed that decreasing the c-stoichiometry could increase the energy conversion efficiency of photosynthesis. Indeed, there is strong evidence that the high H+/ATP of the chloroplast ATP synthase results in a low ATP/nicotinamide adenine dinucleotide phosphate (NADPH) ratio produced by photosynthetic linear electron flow, requiring secondary processes such as cyclic electron flow to support downstream metabolism. We hypothesize that the larger c subunit stoichiometry observed in photosynthetic ATP synthases was selected for because it allows the thylakoid to maintain pmf in a range where ATP synthesis is supported, but avoids excess Δψ and ΔpH, both of which can lead to production of reactive oxygen species and subsequent photodamage. Numerical kinetic simulations of the energetics of chloroplast photosynthetic reactions with altered c-ring size predicts the energy storage of pmf and its effects on the photochemical reaction centers strongly support this hypothesis, suggesting that, despite the low efficiency and suboptimal ATP/NADPH ratio, a high H+/ATP is favored to avoid photodamage. This has important implications for the evolution and regulation of photosynthesis as well as for synthetic biology efforts to alter photosynthetic efficiency by engineering the ATP synthase.

The microbial transfer of electrons to extracellularly located solid compounds, termed extracellular electron transport (EET), is critical for microbial electrode catalysis. Although the components of the EET pathway in the outer membrane (OM) have been identified, the role of electron/cation coupling in EET kinetics is poorly understood. We studied the dynamics of proton transport associated with EET in an OM flavocytochrome complex in Shewanella oneidensis MR‐1. Using a whole‐cell electrochemical assay, a significant kinetic isotope effect (KIE) was observed following the addition of deuterated water (D2O). The removal of a flavin cofactor or key components of the OM flavocytochrome complex significantly increased the KIE in the presence of D2O to values that were significantly larger than those reported for proton channels and ATP synthase, thus indicating that proton transport by OM flavocytochrome complexes limits the rate of EET.

Respiratory complex I couples electron transfer between NADH and ubiquinone to proton translocation across an energy-transducing membrane to support the proton-motive force that drives ATP synthesis. The proton-pumping stoichiometry of complex I (i.e. the number of protons pumped for each two electrons transferred) underpins all mechanistic proposals. However, it remains controversial and has not been determined for any of the bacterial enzymes that are exploited as model systems for the mammalian enzyme. Here, we describe a simple method for determining the proton-pumping stoichiometry of complex I in inverted membrane vesicles under steady-state ADP-phosphorylating conditions. Our method exploits the rate of ATP synthesis, driven by oxidation of NADH or succinate with different sections of the respiratory chain engaged in catalysis as a proxy for the rate of proton translocation and determines the stoichiometry of complex I by reference to the known stoichiometries of complexes III and IV. Using vesicles prepared from mammalian mitochondria (from Bos taurus) and from the bacterium Paracoccus denitrificans, we show that four protons are pumped for every two electrons transferred in both cases. By confirming the four-proton stoichiometry for mammalian complex I and, for the first time, demonstrating the same value for a bacterial complex, we establish the utility of P. denitrificans complex I as a model system for the mammalian enzyme. P. denitrificans is the first system described in which mutagenesis in any complex I core subunit may be combined with quantitative proton-pumping measurements for mechanistic studies.

The flagellated bacterium Escherichia coli is increasingly used experimentally as a self-propelled swimmer. To obtain meaningful, quantitative results that are comparable between different laboratories, reproducible protocols are needed to control, `tune' and monitor the swimming behaviour of these motile cells. We critically review the knowledge needed to do so, explain methods for characterising the colloidal and motile properties of E.coli, cells, and propose a protocol for keeping them swimming at constant speed at finite bulk concentrations. In the process of establishing this protocol, we use motility as a high-throughput probe of aspects of cellular physiology via the coupling between swimming speed and the proton motive force., 18 pages, 16 figures, 4 tables

Contains fulltext : 172525.pdf (Publisher’s version ) (Closed access) Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10(5) molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize all-trans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a non-pumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.

An electrochemical gradient of protons, or proton motive force (PMF), is at the basis of bacterial energetics. It powers vital cellular processes and defines the physiological state of the cell. Here, we use an electric circuit analogy of an Escherichia coli cell to mathematically describe the relationship between bacterial PMF, electric properties of the cell membrane, and catabolism. We combine the analogy with the use of bacterial flagellar motor as a single-cell "voltmeter" to measure cellular PMF in varied and dynamic external environments (for example, under different stresses). We find that butanol acts as an ionophore and functionally characterize membrane damage caused by the light of shorter wavelengths. Our approach coalesces noninvasive and fast single-cell voltmeter with a well-defined mathematical framework to enable quantitative bacterial electrophysiology.

The emergence of multi- and extensively drug resistant tuberculosis worldwide poses a great threat to human health and highlight the need to discover and develop new, effective and inexpensive antituberculosis agents. High-throughput screening assays against well-validated drug targets and structure based drug design have been employed to discover new lead compounds. However, the great majority fail to demonstrate any antimycobacterial activity when tested against Mycobacterium tuberculosis in whole-cell screening assays. This is mainly due to some of the intrinsic properties of the bacilli, such as the extremely low permeability of its cell wall, slow growth, drug resistance, drug tolerance, and persistence. In this sense, understanding the pathways involved in M. tuberculosis drug tolerance, persistence, and pathogenesis, may reveal new approaches for drug development. Moreover, the need for compounds presenting a novel mode of action is of utmost importance due to the emergence of resistance not only to the currently used antituberculosis agents, but also to those in the pipeline. Cheminformatics studies have shown that drugs endowed with antituberculosis activity have the peculiarity of being more lipophilic than many other antibacterials, likely because this leads to improved cell penetration through the extremely waxy mycobacterial cell wall. Moreover, the interaction of the lipophilic moiety with the membrane alters its stability and functional integrity due to the disruption of the proton motive force, resulting in cell death. When a ligand-based medicinal chemistry campaign is ongoing, it is always difficult to predict whether a chemical modification or a functional group would be suitable for improving the activity. Nevertheless, in the “instruction manual” of medicinal chemists, certain functional groups or certain physicochemical characteristics (i.e., high lipophilicity) are considered red flags to look out for in order to safeguard drug-likeness and avoid attritions in the drug discovery process. In this review, we describe how antituberculosis compounds challenge established rules such as the Lipinski's “rule of five” and how medicinal chemistry for antituberculosis compounds must be thought beyond such dogmatic schemes.

Gram-negative bacteria import essential nutrients such as iron and vitamin B12 through outer membrane receptors. This process utilizes proton motive force harvested by the Ton system made up of three inner membrane proteins, ExbB, ExbD and TonB. ExbB and ExbD form the proton channel that energizes uptake through TonB. Recently, crystal structures suggest that the ExbB pentamer is the scaffold. Here, we present structures of hexameric complexes of ExbB and ExbD revealed by X-ray crystallography and single particle cryo-EM. Image analysis shows that hexameric and pentameric complexes coexist, with the proportion of hexamer increasing with pH. Channel current measurement and 2D crystallography support the existence and transition of the two oligomeric states in membranes. The hexameric complex consists of six ExbB subunits and three ExbD transmembrane helices enclosed within the central channel. We propose models for activation/inactivation associated with hexamer and pentamer formation and utilization of proton motive force., eLife digest Many biological processes that are essential for life are powered by the flow of ions across the membranes of cells. Similar to how energy is stored in the water behind a dam, energy is also stored when the concentration of ions on one side of a biological membrane is higher than it is on the other. When these ions then flow down this concentration gradient, the energy can be harnessed to power other processes. In many bacteria, the concentration of hydrogen ions, or protons, is higher on the outside of the cell. When the protons flow down the concentration gradient, a protein complex called the Ton system in the bacteria’s inner membrane harnesses the energy to transport various compounds, including essential nutrients, across the outer membrane, which is about 20 nanometres away. Toxins, and viruses that infect bacteria, can also hijack the Ton system to gain entry into these cells. This means that the Ton system could perhaps be targeted via drugs to treat bacterial infections. Though the Ton system is important, structural information on this protein family is limited. The Ton complex is composed of three proteins – ExbB, ExbD and TonB – located in the bacteria’s inner membrane. ExbB and ExbD together form a channel for the protons and the complex made from these two proteins can be thought of as the system’s engine. Maki-Yonekura et al. wanted to understand how the ExbB / ExbD complex works, which was challenging because the complex was not well suited to any single structural biology technique. To get around this issue, a combination of two techniques called X-ray crystallography and single particle cryo-EM were used. This approached revealed that the two proteins form complexes made up of either five or six ExbB subunits with one or three ExbD subunits, respectively. It also showed that the proteins transition between the two forms in a cell’s membrane. More of the larger six-unit complex (also called a “hexamer”) formed at higher pH. This is consistent with the increased flow of protons through the channel when the local conditions inside the cell become less acidic. Based on these results, Maki-Yonekura et al. propose that some subunits in the core of the complex rotate to harness the energy from the flow of protons, and the number of subunits in the complex changes when it switches to become active or inactive. The discoveries may provide a new vision of dynamic membrane biology. Further studies are now needed to see how general this mechanism is in biology, and the new structural information could also be used to help develop more anti-bacterial drugs.

In addition to ∆pH formed across the thylakoid membrane, membrane potential contributes to proton motive force (pmf) in chloroplasts. However, the regulation of photosynthetic electron transport is mediated solely by ∆pH. To assess the contribution of two cyclic electron transport pathways around photosystem I (one depending on PGR5/PGRL1 and one on NDH) to pmf formation, electrochromic shift (ECS) was analyzed in the Arabidopsis pgr5 mutant, NDH-defective mutants (ndhs and crr4-2), and their double mutants (ndhs pgr5 and crr4-2 pgr5). In pgr5, the size of the pmf, as represented by ECSt, was reduced by 30% to 47% compared with that in the wild type (WT). A gH+ parameter, which is considered to represent the activity of ATP synthase, was enhanced at high light intensities. However, gH+ recovered to its low-light levels after 20min in the dark, implying that the elevation in gH+ is due to the disturbed regulation of ATP synthase rather than to photodamage. After long dark adaptation more than 2h, gH+ was higher in pgr5 than in the WT. During induction of photosynthesis, gH+ was more rapidly elevated in pgr5 than that in the WT. Both results suggest that ATP synthase is not fully inactivated in the dark in pgr5. In the NDH-deficient mutants, ECSt was slightly but significantly lower than in the WT, whereas gH+ was not affected. In the double mutants, ECSt was even lower than in pgr5. These results suggest that both PGR5/PGRL1- and NDH-dependent pathways contribute to pmf formation, although to different extents. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

Although photoinhibition of photosystem II (PSII) frequently occurs under natural growing conditions, knowledge about the effect of moderate photoinhibition on linear electron flow (LEF) remains controversial. Furthermore, mechanisms underlying the decrease in LEF upon PSII photoinhibition are not well clarified. We examined how selective PSII photoinhibition influenced LEF in the attached leaves of shade-demanding plant Panax notoginseng. After leaves were exposed to a high level of light (2258 μmol photons m-2 s-1) for 30 and 60 min, the maximum quantum yield of PSII (Fv/Fm) decreased by 17 and 23%, respectively, whereas the maximum photo-oxidizable P700 (Pm) remained stable. Therefore, this species displayed selective PSII photodamage under strong illumination. After these treatments, LEF was significantly decreased under all light levels but acidification of the thylakoid lumen changed only slightly. Furthermore, the decrease in LEF under low light was positively correlated with the extent of PSII photoinhibition. Thus, the decline in LEF was not caused by the enhancement of lumenal acidification, but was induced by a decrease in PSII activity. These results indicate that residual PSII activity is an important determinant of LEF in this shade-adapted species, and they provide new insight into how strong illumination affects the growth of shade-demanding plants.

The Rcs phosphorelay and Psp (phage shock protein) systems are envelope stress responses that are highly conserved in gammaproteobacteria. The Rcs regulon was found to be strongly induced during metal deprivation of Salmonella enterica serovar Typhimurium lacking the Psp response. Nineteen genes activated by the RcsA-RcsB response regulator make up an operon responsible for the production of colanic acid capsular polysaccharide, which promotes biofilm development. Despite more than half a century of research, the physiological function of colanic acid has remained elusive. Here we show that Rcs-dependent colanic acid production maintains the transmembrane electrical potential and proton motive force in cooperation with the Psp response. Production of negatively charged exopolysaccharide covalently bound to the outer membrane may enhance the surface potential by increasing the local proton concentration. This provides a unifying mechanism to account for diverse Rcs/colanic acid-related phenotypes, including susceptibility to membrane-damaging agents and biofilm formation., IMPORTANCE Colanic acid is a negatively charged polysaccharide capsule produced by Escherichia coli, Salmonella, and other gammaproteobacteria. Research conducted over the 50 years since the discovery of colanic acid suggests that this exopolysaccharide plays an important role for bacteria living in biofilms. However, a precise physiological role for colanic acid has not been defined. In this study, we provide evidence that colanic acid maintains the transmembrane potential and proton motive force during envelope stress. This work provides a new and fundamental insight into bacterial physiology.

There is considerable interest in improving plant productivity by altering the dynamic responses of photosynthesis in tune with natural conditions. This is exemplified by the ‘energy-dependent' form of non-photochemical quenching ( q E ), the formation and decay of which can be considerably slower than natural light fluctuations, limiting photochemical yield. In addition, we recently reported that rapidly fluctuating light can produce field recombination-induced photodamage (FRIP), where large spikes in electric field across the thylakoid membrane (Δ ψ ) induce photosystem II recombination reactions that produce damaging singlet oxygen ( 1 O 2 ). Both q E and FRIP are directly linked to the thylakoid proton motive force ( pmf ), and in particular, the slow kinetics of partitioning pmf into its ΔpH and Δ ψ components. Using a series of computational simulations, we explored the possibility of ‘hacking' pmf partitioning as a target for improving photosynthesis. Under a range of illumination conditions, increasing the rate of counter-ion fluxes across the thylakoid membrane should lead to more rapid dissipation of Δ ψ and formation of ΔpH. This would result in increased rates for the formation and decay of q E while resulting in a more rapid decline in the amplitudes of Δ ψ -spikes and decreasing 1 O 2 production. These results suggest that ion fluxes may be a viable target for plant breeding or engineering. However, these changes also induce transient, but substantial mismatches in the ATP : NADPH output ratio as well as in the osmotic balance between the lumen and stroma, either of which may explain why evolution has not already accelerated thylakoid ion fluxes. Overall, though the model is simplified, it recapitulates many of the responses seen in vivo , while spotlighting critical aspects of the complex interactions between pmf components and photosynthetic processes. By making the programme available, we hope to enable the community of photosynthesis researchers to further explore and test specific hypotheses. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’.

The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ∼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell. DOI: http://dx.doi.org/10.7554/eLife.23136.001, eLife digest Most bacteria are able to move in a directed manner towards nutrients or other locations of interest. Many move by rotating long tail-like filaments called flagella that stick out from the cell. The flagellum is a remarkably complex nanomachine. It is several times longer than the main body of the bacterial cell body and its external filament is made of thousands of building blocks of a single protein called flagellin. This protein is made inside the cell and a structure at the base of the flagellum known as a type III secretion system uses chemical energy to pump it out of the cell so that it can be incorporated into the growing flagellum. The exported building blocks travel through a narrow channel within the flagellum and self-assemble at the tip. It has been a mystery for several decades how bacteria manage to assemble the building blocks of flagella outside of the cell, where no discernible energy source is available. Renault et al. used mathematical modeling, biochemical and microscopy techniques to observe how the flagella of a bacterium called Salmonella enterica assemble in real time. The experiments demonstrate that simple biophysical principles regulate the assembly of the flagellum. The building blocks are pumped into the channel of the flagellum by the type III secretion system and then diffuse to the tip of the filament. Accordingly, the longer the flagellum gets, the slower it grows. This molecular mechanism also explains why the growth of bacterial flagella will eventually stop even without any other control mechanisms in place. Further work will be needed to understand how the type III secretion system harnesses chemical energy to drive the movement of flagellin out of the cell into the growing flagellum. A molecular understanding of these processes will aid the design of new antibiotics targeted against type III secretion systems. DOI: http://dx.doi.org/10.7554/eLife.23136.002

For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly–disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Outer-membrane c-type cytochrome (OM c-Cyt) complexes in several genera of iron-reducing bacteria, such as Shewanella and Geobacter, are capable of transporting electrons from the cell interior to extracellular solids as a terminal step of anaerobic respiration. The kinetics of this electron transport has implications for controlling the rate of microbial electron transport during bioenergy or biochemical production, iron corrosion, and natural mineral cycling. Herein, we review the findings from in-vivo and in-vitro studies examining electron transport kinetics through single OM c-Cyt complexes in Shewanella oneidensis MR-1. In-vitro electron flux via a purified OM c-Cyt complex, comprised of MtrA, B, and C proteins from S. oneidensis MR-1, embedded in a proteoliposome system is reported to be 10- to 100-fold faster compared with in-vivo estimates based on measurements of electron flux per cell and OM c-Cyts density. As the proteoliposome system is estimated to have 10-fold higher cation flux via potassium channels than electrons, we speculate that the slower rate of electron-coupled cation transport across the OM is responsible for the significantly lower electron transport rate that is observed in-vivo. As most studies to date have primarily focused on the energetics or kinetics of interheme electron hopping in OM c-Cyts in this microbial electron transport mechanism, the proposed model involving cation transport provides new insight into the rate detemining step of EET, as well as the role of self-secreted flavin molecules bound to OM c-Cyt and proton management for energy conservation and production in S. oneidensis MR-1.

During photosynthesis, photosynthetic electron transport generates a proton motive force (pmf) across the thylakoid membrane, which is used for ATP biosynthesis via ATP synthase in the chloroplast. The pmf is composed of an electric potential (ΔΨ) and an osmotic component (ΔpH). Partitioning between these components in chloroplasts is strictly regulated in response to fluctuating environments. However, our knowledge of the molecular mechanisms that regulate pmf partitioning is limited. Here, we report a bestrophin‐like protein (AtBest), which is critical for pmf partitioning. While the ΔpH component was slightly reduced in atbest, the ΔΨ component was much greater in this mutant than in the wild type, resulting in less efficient activation of nonphotochemical quenching (NPQ) upon both illumination and a shift from low light to high light. Although no visible phenotype was observed in the atbest mutant in the greenhouse, this mutant exhibited stronger photoinhibition than the wild type when grown in the field. AtBest belongs to the bestrophin family proteins, which are believed to function as chloride (Cl−) channels. Thus, our findings reveal an important Cl− channel required for ion transport and homeostasis across the thylakoid membrane in higher plants. These processes are essential for fine‐tuning photosynthesis under fluctuating environmental conditions.

Background: ATP synthesis is driven by the combination of transmembrane electrical potential and pH difference. Results: Either electrical potential or pH difference can drive synthesis even when the other opposes. Conclusion: The synthesis rate depends on the algebraic sum of the two, irrespective of the individual magnitudes and signs. Significance: Comprehensive data sets directly attest to kinetic equivalence of the two., ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (ΔpH) across the membrane and transmembrane electrical potential (Δψ). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted, and previous experimental results vary. Here, we show that with the thermophilic Bacillus PS3 ATP synthase that lacks an inhibitory domain of the ϵ subunit, ΔpH imposed by acid-base transition and Δψ produced by valinomycin-mediated K+ diffusion potential contribute equally to the rate of ATP synthesis within the experimental range examined (ΔpH −0.3 to 2.2, Δψ −30 to 140 mV, pH around the catalytic domain 8.0). Either ΔpH or Δψ alone can drive synthesis, even when the other slightly opposes. Δψ was estimated from the Nernst equation, which appeared valid down to 1 mm K+ inside the proteoliposomes, due to careful removal of K+ from the lipid.

In bacteria, the major route of protein translocation across the cytoplasmic membrane is the so-called “Sec-pathway”. Secretory proteins that use this route are synthesized as precursors (preproteins) with an amino-terminal extension, the signal peptide. The signal peptide consists of three domains: a positively charged N-terminus (N-domain), a hydrophobic central region (H-domain) and a C-terminal domain containing the signal peptidase cleavage site. The signal peptide directs the preprotein to the translocation machinery and is needed for initiation of the translocation process. It also slows down the folding of the mature domain of a secretory preprotein, and thereby increases the time window for molecular chaperones like SecB to interact. SecB is a molecular chaperone that stabilizes preproteins in a translocation-competent (non-aggregated, loosely folded) state and that targets them to the membrane (Fig. 1, route b) (reviewed by Fekkes and Driessen, 1999). Preproteins with a very hydrophobic signal peptide and most membrane proteins are targeted to the cytoplasmic (inner) membrane via the signal recognition particle (SRP)-pathway (Fig. 1, route c). SRP binds to the H-domain of the signal peptide when it emerges from the ribosome, and subsequently targets the ribosome-bound nascent chain complex via the SRP receptor FtsY at the membrane to the translocation machinery. Preprotein translocation is mediated by a multicomponent membrane complex that consists of the membrane-associated ATPase SecA, and a large integral membrane domain consisting of SecY, SecE, SecG, SecD, SecF and YajC as separate subunits.

Monoderm bacteria possess a cell envelope made of a cytoplasmic membrane and a cell wall, whereas diderm bacteria have and extra lipid layer, the outer membrane, covering the cell wall. Both cell types can also produce extracellular protective coats composed of polymeric substances like, for example, polysaccharidic capsules. Many of these structures form a tight physical barrier impenetrable by phage virus particles. Tailed phages evolved strategies/functions to overcome the different layers of the bacterial cell envelope, first to deliver the genetic material to the host cell cytoplasm for virus multiplication, and then to release the virion offspring at the end of the reproductive cycle. There is however a major difference between these two crucial steps of the phage infection cycle: virus entry cannot compromise cell viability, whereas effective virion progeny release requires host cell lysis. Here we present an overview of the viral structures, key protein players and mechanisms underlying phage DNA entry to bacteria, and then escape of the newly-formed virus particles from infected hosts. Understanding the biological context and mode of action of the phage-derived enzymes that compromise the bacterial cell envelope may provide valuable information for their application as antimicrobials.

Sec translocase catalyzes membrane protein insertion and translocation. We have introduced stretches of charged amino acid residues into the preprotein proOmpA and have analyzed their effect on in vitro protein translocation into Escherichia coli inner membrane vesicles. Both negatively and positively charged amino acid residues inhibit translocation of proOmpA, yielding a partially translocated polypeptide chain that blocks the translocation site and no longer activates preprotein-stimulated SecA ATPase activity. Stretches of positively charged residues are much stronger translocation inhibitors and suppressors of the preprotein-stimulated SecA ATPase activity than negatively charged residues. These results indicate that both clusters of positively and negatively charged amino acids are poor substrates for the Sec translocase and that this is reflected by their inability to stimulate the ATPase activity of SecA. (C) 2009 Elsevier Ltd. All rights reserved.

About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they are targeted to the Sec translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The Sec translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research.

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 microM increase F(V) as efficiently as 100 microM of spermidine or 1000 microM of putrescine or 1000 microM of Mg(2+). It is also demonstrated that the increase in F(V) derives mainly from the contribution of PSIIalpha centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.

Multidrug resistance (MDR) has appeared in response to selective pressures resulting from the incorrect use of antibiotics and other antimicrobials. This inappropriate application and mismanagement of antibiotics have led to serious problems in the therapy of infectious diseases. Bacteria can develop resistance by various mechanisms and one of the most important factors resulting in MDR is efflux pump-mediated resistance. Because of the importance of the efflux-related multidrug resistance the development of new therapeutic approaches aiming to inhibit bacterial efflux pumps is a promising way to combat bacteria having over-expressed MDR efflux systems. The definition of an efflux pump inhibitor (EPI) includes the ability to render the bacterium increasingly more sensitive to a given antibiotic or even reverse the multidrug resistant phenotype. In the recent years numerous EPIs have been developed, although so far their clinical application has not yet been achieved due to their in vivo toxicity and side effects. In this review, we aim to give a short overview of efflux mediated resistance in bacteria, EPI compounds of plant and synthetic origin, and the possible methods to investigate and screen EPI compounds in bacterial systems.

International audience; BACKGROUND: Limitations in treatment of biofilm-associated bacterial infections are often due to subpopulation of persistent bacteria (persisters) tolerant to high concentrations of antibiotics. Based on the increased aminoglycoside efficiency under alkaline conditions, we studied the combination of gentamicin and the clinically compatible basic amino acid L-arginine against planktonic and biofilm bacteria both in vitro and in vivo. METHODS: Using Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli bioluminescent strains, we studied the combination of L-arginine and gentamicin against planktonic persisters through time-kill curves of late stationary-phase cultures. In vitro biofilm tolerance towards gentamicin was assessed using PVC 96 well-plates assays. Efficacy of gentamicin as antibiotic lock treatment (ALT) at 5 mg/mL at different pH was evaluated in vivo using a model of totally implantable venous access port (TIVAP) surgically implanted in rats. RESULTS: We demonstrated that a combination of gentamicin and the clinically compatible basic amino acid L-arginine increases in vitro planktonic and biofilm susceptibility to gentamicin, with 99% mortality amongst clinically relevant pathogens, i.e. S. aureus, E. coli and P. aeruginosa persistent bacteria. Moreover, although gentamicin local treatment alone showed poor efficacy in a clinically relevant in vivo model of catheter-related infection, gentamicin supplemented with L-arginine led to complete, long-lasting eradication of S. aureus and E. coli biofilms, when used locally. CONCLUSION: Given that intravenous administration of L-arginine to human patients is well tolerated, combined use of aminoglycoside and the non-toxic adjuvant L-arginine as catheter lock solution could constitute a new option for the eradication of pathogenic biofilms.

Polyamines (PAs) are low molecular weight amines that occur in every living organism. The three main PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)] are involved in several important biochemical processes covered in recent reviews. As rule of thumb, increase of the cellular titer of PAs in plants is related to cell growth and cell tolerance to abiotic and biotic stress. In the present contribution, we describe recent findings from plant bioenergetics that bring to light a previously unrecognized dynamic behavior of the PA pool. Traditionally, PAs are described by many authors as organic polycations, when in fact they are bases that can be found in a charged or uncharged form. Although uncharged forms represent less than 0.1% of the total pool, we propose that their physiological role could be crucial in chemiosmosis. This process describes the formation of a PA gradient across membranes within seconds and is difficult to be tested in vivo in plants due to the relatively small molecular weight of PAs and the speed of the process. We tested the hypothesis that PAs act as permeable buffers in intact leaves by using recent advances in vivo probing. We found that an increase of PAs increases the electric component (∆ψ) and decreases the ∆pH component of the proton motive force (pmf). These findings reveal an important modulation of the energy production process and photoprotection of the chloroplast by PAs. We explain in detail the theory behind PA pumping and ion trapping in acidic compartments (such as the lumen in chloroplasts) and how this regulatory process could improve either the photochemical efficiency of the photosynthetic apparatus and increase the synthesis of ATP or fine tune antenna regulation and make the plant more tolerant to stress.

Fundamental studies on the availability of oxygen from the decomposition of H(2)O(2), in vivo, by Xanthomonas campestris, when H(2)O(2) is used as an oxygen source are presented. It was found that the H(2)O(2) added extracellularly (0.1-6 mM) was decomposed intracellularly. Further, when H(2)O(2) was added, the flux of H(2)O(2) into the cell, is regulated by the cell. The steady-state H(2)O(2) flux into the cell was estimated to be 9.7 x 10(-8) mol m(-2) s(-1). In addition, it was proved that the regulation of H(2)O(2) flux was coupled to the protonmotive force (PMF) using experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which disrupts PMF. The coupling constant between the rate of free energy availability from PMF and the rate of reduction of H(2)O(2) flux, was found to be 46.4 mol m(-2) s(-1) J(-1) from simulations using a developed model. Also, the estimated periplasmic catalase concentration was 1.4 x 10(-9) M.

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.

Amino acid transport in Lactobacillus helveticus was analyzed. Strain specificity of amino acid transport was speculated between L. helveticus NCDO2712 and SBT2171. Glucose energized L. helveticus NCDO2712 actively transported and accumulated the essential and growth stimulating amino acids (leucine, isoleucine, valine, threonine, lysine, aspartic acid, glutamic acid, tyrosine, arginine, and histidine). Uptake of proline, phenylalanine and tryptophan was not observed. Transport studies in isolated membrane vesicles of L. helveticus fused with liposomes to reduce the passive proton permeability revealed that amino acids leucine, isoleucine, valine, threonine, and lysine are transported by a proton motive force coupled system. These results indicated that L. helveticus and Lactococcus lactis amino acid transport systems are similar.

The homodimeric SecA protein is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade. SecA contains two essential nucleotide binding sites (NBSs), i.e., NBS1 and NBS2 that hind ATP with high and low affinity, respectively. The photoactivatable bifunctional cross-linking agent 3'-arylazido-8-azidoadenosine 5'-triphosphate (diN(3)ATP) was used to investigate the spatial arrangement of the nucleotide binding sites of SecA, DiN(3)ATP is an authentic ATP analogue as it supports SecA-dependent precursor protein translocation and translocation ATPase, UV-induced photo-cross-linking of the diN(3)ATP-bound SecA results in the formation of stable dimeric species of SecA. D209N SecA, a mutant unable to bind nucleotides at NBS1, was also photo-crosslinked by diN(3)ATP, whereas no cross-linking occurred with the NBS2 mutant R509K SecA, We concluded that the low-affinity NBS2, which is located in the carboxyl-terminal half of SecA, Is the site of crosslinking and that NBS2 binds nucleotides at or near the subunit interface of the SecA dimer.

Lactococcus lactis can undergo respiration when hemin is added to an aerobic culture. The most distinctive feature of lactococcal respiration is that lactate could be consumed in the stationary phase concomitantly with the rapid accumulation of diacetyl and acetoin. However, the enzyme responsible for lactate utilization in this process has not yet been identified. As genes for fermentative NAD-dependent l-lactate dehydrogenase (l-nLDH) and potential electron transport chain (ETC)-related NAD-independent l-LDH (l-iLDH) exist in L. lactis, the activities of these enzymes were measured in this study using crude cell extracts prepared from respiratory and fermentation cultures. Further studies were conducted with purified preparations of recombinant LDH homologous proteins. The results showed that l-iLDH activity was hardly detected in both crude cell extracts and purified l-iLDH homologous protein while l-nLDH activity was very significant. This suggested that l-iLDHs were inactive in lactate utilization. The results of kinetic analyses and the effects of activator, inhibitor, substrate and product concentrations on the reaction equilibrium showed that l-nLDH was much more prone to catalyze the pyruvate reduction reaction but could reverse its role provided that the concentrations of NADH and pyruvate were extremely low while NAD and lactate were abundant. Metabolite analysis in respiratory culture revealed that the cellular status in the stationary phase was beneficial for l-nLDH to catalyze lactate oxidation. The factors accounting for the respiration- and stationary phase-dependent lactate utilization in L. lactis are discussed here., Highlights • LutABC proteins do not participate in lactate oxidation in Lactococcus lactis • Lactococcus lactis has very low NAD-independent lactate dehydrogenase activity • Fructose-1,6-bisphosphate-dependent lactate dehydrogenase can work in reverse in vivo • Metabolite concentrations in the stationary phase are favorable for lactate oxidation • Respiratory metabolism is the basis for continual lactate oxidation in Lactococcus

Rnf is a membrane protein complex that has been shown to be important in energy conservation. Here,Desulfovibrio alaskensisG20 and Rnf mutants of G20 were grown with different electron donor and acceptor combinations to determine the importance of Rnf in energy conservation and the type of ion gradient generated. The addition of the protonophore TCS strongly inhibited lactate-sulfate dependent growth whereas the sodium ionophore ETH2120 had no effect, indicating a role for the proton gradient during growth. Mutants inrnfAandrnfDwere more sensitive to the protonophore at 5 µM than the parental strain, suggesting the importance of Rnf in the generation of a proton gradient. The electrical potential (ΔΨ), ΔpH and proton motive force were lower in thernfAmutant than in the parental strain ofD.alaskensisG20. These results provide evidence that the Rnf complex inD. alaskensisfunctions as a primary proton pump whose activity is important for growth.

(1994). Cellular Toxicity of Lipophilic Compounds: Mechanisms, Implications, and Adaptations. Biocatalysis: Vol. 10, No. 1-4, pp. 113-122.